Phytoestrogen product of red clover and pharmaceutical uses thereof

ABSTRACT

The present invention provides compositions comprising optimized ratios of Red clover phytoestrogens as determined by a proprietary physiologically based pharmacokinetic and pharmacodynamic model. The compositions are useful for modulating, preventing or treating postmenopausal or climacteric symptoms, which include but are not limited to bone loss, bone remodeling, hot flushes and vaginal atrophy. The present invention also provides methods for modulating, preventing or treating postmenopausal or climacteric symptoms using the compositions disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 14/242,981, filed Apr. 2, 2014, which is acontinuation-in-part application of International application No.PCT/IB2012/055277, filed Oct. 2, 2012, which claims benefit of U.S.App'l No. 61/542,253, filed Oct. 2, 2011; U.S. application Ser. No.14/242,981 also is a continuation-in-part application of U.S.application Ser. No. 14/069,740, filed Nov. 1, 2013, which is acontinuation of U.S. application Ser. No. 13/251,267, filed Oct. 2,2011, which is a continuation-in-part of U.S. application Ser. No.13/028,136, filed Feb. 15, 2011, which claims the benefit of U.S. App'lNo. 61/304,589, filed Feb. 15, 2010. The entire contents of thepreceding applications are incorporated herein by reference into thisapplication.

BACKGROUND OF THE INVENTION

Deficiency of estrogens during menopause can lead to a number ofcomplications including hot flushes, reduced bone density, mood swings,vaginal atrophy, etc. These symptoms are commonly treated with synthetichormones. Although postmenopausal symptoms can be alleviated, hormonereplacement therapy (HRT) was discovered to be associated with increasedcardiovascular disorders in one of the largest studies of its kind(Women's health Initiative, WHI) (Seelig, Altura et al. 2004). HRT wasalso linked to increased risk of breast and ovarian cancer (Fernandez,Gallus et al. 2003, Gambacciani, Monteleone et al. 2003). After the WHItrial results were published, the use of HRT was reduced dramatically.Many postmenopausal women have resorted to alternative therapy becausephytoestrogens are generally considered to be safe and efficacious. Theuse of soy and Red clover; the most common species used is Trifoliumpratense, which are rich in phytoestrogens, has been on the rise (Beck,Rohr et al. 2005). Despite the trend, clinical trial results onphytoestrogens, however, have been equivocal. For example, the effectsof Red clover on postmenopausal bone loss have been marginal (Beck, Rohret al. 2005, Booth, Piersen et al. 2006, Wuttke, Jarry et al. 2007, Ma,Qin et al. 2008, Lagari and Levis 2014). Despite occasional success, alarge percentage of clinical trials showed no significant effects onpostmenopausal bone loss. Similar data have been obtained for otherpostmenopausal symptoms such as hot flashes (Ghazanfarpour, Sadeghi etal. 2015, Ghazanfarpour, Sadeghi et al. 2016) and vaginal atrophy(Ghazanfarpour, Latifnejad Roudsari et al. 2015, Ghazanfarpour, Sadeghiet al. 2016). Alternative therapy has not replaced HRT effectively. Arecent study showed that the trend of women moving away from HRT has ledto an alarming increase in bone fractures and it is estimated thatfractures related to menopause is expected to exceed 40,000 per year inwomen aged 65-69 years (Gambacciani, Ciaponi et al. 2007). Since theside effects of HRT were publicized after the WHI trial, it has sincebeen reevaluated. There is no consensus with regard to HRT's safetyamong the medical research community. Therefore, a much closer look atthe ‘less than expected’ effects of phytoestrogens should be undertakenbecause the toxicity profile of this type of products is so much morefavorable.

Besides HRT, there are no drugs that could be used effectively fortreating climacteric symptoms such as hot flashes and vaginal atrophy.Anti-depressants like Prozac, Paxil or Effexor, blood pressuremedications like clonidine, anti-seizure drugs like gabapentin, and aparoxetine formula Brisdelle have been used for treating hot flashes.However, these drugs produce significant side effects, and the efficacyis at best marginal. Ospemifene, a selective estrogen receptormodulator, has been approved for the treatment of vaginal atrophy.However, its long term safety has not been ascertained. Genistein, a soyisoflavone has been shown to have some positive effects on vaginal cellstructure.

The major bioactive isoflavones in soy are genistein, daidzein,glycitein and prunetin (Setchell and Cassidy 1999). They are alsopresent in their glycoside forms. There are three classes of bioactivesin red clover: isoflavones, coumestrols and lignans (Beck, Rohr et al.2005). The quantity of coumestrols and lignans is small; therefore,their contribution to the overall activity is likely minimal. The majorisoflavones in red clover are Biochanin A and formononetin (Liu,Burdette et al. 2001, Overk, Yao et al. 2005, Booth, Overk et al. 2006).Genistein and daidzein are present in minute quantities. Biochanin A andformononetin are precursors of their respective active moieties,genistein and daidzein. The conversion takes place in the intestine byintestinal flora and liver, although the relative significance has notbeen established. Daidzein is converted by bacteria in the colon to forma more estrogenic metabolite, equol. In Red clover, a significantquantity of Biochanin A and formononetin is in the form of glycosides.The glycosides in soy and red clover are converted to their respectiveaglycones by the intestinal flora before absorption (Setchell andCassidy 1999).

Relative absorption of isoflavone glycoside and their respectiveaglycones is a subject of controversy. Although the cause of controversyis not readily apparent, the low solubility of the aglycones in apreparation may have a profound effect on their dissolution, metabolismand absorption.

Formononetin and Biochanin A are de-methylated by the intestinal microflora to produce two active metabolites daidzein and genistein,respectively (Hur and Rafii 2000). However, the site of this metabolicpathway is questioned (Tolleson, Doerge et al. 2002).

Metabolism of isoflavones is mainly mediated by Phase II enzymes in theenterocytes and hepatocytes. Although metabolism of individualisoflavones in rats has been well characterized (Jia, Chen et al. 2004,Chen, Lin et al. 2005, Chen, Wang et al. 2005), interaction betweencomponents has not been evaluated.

Clinical studies showed that extracts of red clover or soy are safe;however, their efficacies are equivocal (Booth, Piersen et al. 2006,Lagari and Levis 2014). Although there are proprietary products in themarket, which have shown potentials for treating or preventingpostmenopausal osteoporosis, hot flushes and vaginal atrophy; theseproducts unfortunately, do not have the quality of a drug. The majorshortcomings for the design of these products in the market are thatthey have not taken into consideration of the interplay betweenpharmacokinetics and pharmacodynamics. In other words, proper dosageand/or dosing interval are empirically decided.

In this invention, the interplay between these “active” components isevaluated and quantified using a proprietary physiologically basedpharmacokinetic and pharmacodynamic model (PBPKPD).

One of the objectives of the present invention is to providecompositions comprising active ingredients of Red clover that areeffective in modulating, preventing or treating climacteric symptoms.The compositions provided herein are formulated as special dosage formsand require a much lower dosage of phytoestrogens than Red cloverproducts available in the market. The advantage of the presentcompositions is their consistency. By modifying the mode of delivery,the other advantage of the present compositions is the increase in thebioavailability of the aglycones and eliminates the conversion to theirrespective bioactive metabolites in the colon, which leads tovariability in efficacy.

SUMMARY OF THE INVENTION

The present invention discloses compositions of active ingredients inRed clover, which are optimized to modulate, prevent or treatpostmenopausal or climacteric symptoms, which include but are notlimited to bone loss, bone remodeling, hot flashes and vaginal atrophy.In one embodiment, the present invention provides methods formodulating, preventing or treating postmenopausal or climactericsymptoms using the compositions disclosed herein. In one embodiment, thecomposition comprises at least 80% of Biochanin A, at least 1% ofgenistein, and no more than 5% each of formononetin and daidzein. Inanother embodiment, the composition is formulated as parenteral dosageforms, which include but are not limited to intravenous, intramuscularand subcutaneous delivery. In another embodiment, the composition isformulated as topical dosage forms which include but are not limited totransdermal and vaginal delivery. In another embodiment, the compositionis formulated into sublingual and buccal dosage forms.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 shows a typical LC/MS chromatogram showing the composition of aRed clover extract.

FIG. 2 shows the metabolism of the isoflavone mixtures by human fecalbacteria.

FIG. 3 shows the metabolism of the isoflavone mixtures by humanintestinal microsomes.

FIG. 4 shows the metabolism of the isoflavone mixtures by humanhepatocytes.

FIG. 5 shows the effects of individual Red clover isoflavones onosteoblast differentiation of MC3T3 cells. The total concentration ofisoflavone in each treatment is 10 μM. B: Biochanin A; D: daidzein; E:equol; F: formononetin; G: genistein; and RC: Red clover extract.ALP/cell number ratio obtained in each treatment is normalized by thecontrol to quantify relative osteoblast activities.

FIG. 6 shows the effects of Red clover isoflavone mixtures on osteoblastdifferentiation of MC3T3 cells. The total concentration of isoflavone ineach treatment is 10 μM. For pair treatment, the ratio is 1:9. B isBiochanin A; DB is 1 μM of daidzein and 9 μM of Biochanin A; EB is 1 μMof equol and 9 μM of Biochanin A; FB is 1 μM of formononetin and 9 μM ofBiochanin A; GB is 1 μM of genistein and 9 μM of Biochanin A; and RC isRed clover extract. ALP/cell number ratio obtained in each treatment isnormalized by the control to quantify relative osteoblast activities.

FIG. 7 shows the effects of progressive increase of genistein in amixture of genistein and Biochanin A on the osteoblast differentiationof MC3T3 cells. The total concentration of isoflavone in each treatmentis 10 μM. Effect of Red clover extract is also tested. ALP/cell numberratio obtained in each treatment is normalized by the control toquantify relative osteoblast activities.

FIG. 8 shows the inhibition of osteoclast differentiation by Red cloverisoflavones. The total concentration of isoflavone in each treatment is10 μM. B: Biochanin A; D: daidzein; E: equol; F: formononetin; G:genistein; and RC: Red clover extract. For pair treatment, the ratio is1:9. BD is 1 μM of Biochanin A and 9 μM of daidzein; DB is 1 μM ofdaidzein and 9 μM of Biochanin A; EB is 1 μM of equol and 9 μM ofBiochanin A; FB is 1 μM of formononetin and 9 μM of Biochanin A; GB is 1μM of genistein and 9 μM of Biochanin A; and RC is Red clover extract.ACP/cell number ratio obtained in each treatment is normalized by thecontrol to quantify relative osteoclast activities.

FIG. 9 shows the effects of formononetin on the solubility of BiochaninA in fasted simulated intestinal fluid (FaSSIF). The amount of BiochaninA in the mixture is 200 μg/mL.

FIG. 10 shows the effects of Biochanin A on the solubility offormononetin in fasted simulated intestinal fluid (FaSSIF). The amountof formononetin in the mixture is 50 μg/mL.

FIG. 11 shows the effects of genistein on the solubility of Biochanin Ain fasted simulated intestinal fluid (FaSSIF). The amount of Biochanin Ain the mixture is 200 μg/mL.

FIG. 12 shows the effects of daidzein on the solubility of Biochanin Ain fasted simulated intestinal fluid (FaSSIF). The amount of Biochanin Ain the mixture is 200 μg/mL

FIG. 13 shows the Isoflavone profiles of a commercial Red clover extract(Shaanxi, 40% total phytoestrogen) after sequential extraction withmethanol.

FIG. 14 shows the Isoflavone profiles of a commercial Red clover extract(Acetar, 40% total phytoestrogen) after sequential extraction withmethanol.

FIG. 15 shows a dissolution profile of two PROMENSIL tablets, eachcontains 40 mg of total phytoestrogens. The content of Biochanin A andformononetin was not completely released.

DETAILED DESCRIPTION OF THE INVENTION

Biochanin A and formononetin are the major components in Red clover.Their colonic metabolites, genistein and daidzein are also present inminute quantities (Beck, Rohr et al. 2005). A colonic metabolite ofdaidzein, equol, has been shown to have the highest estrogenicity amongthe red clover phytoestrogens (Magee 2011).

As shown below, the two major aglycones of Red clover, Biochanin A andformononetin are highly insoluble in the gastrointestinal fluids.Daidzein and genistein, two minute components in Red clover, are alsoinsoluble, although they are more soluble than Biochanin A andformononetin.

In the present invention, Biochanin A, formononetin, daidzein andgenistein are found interacting with each other at the solubility level.The presence of one phytoestrogen may enhance or inhibit the solubilityof the other phytoestrogen.

Lack of solubility, high first-pass gut and liver metabolism and colonicbacteria metabolism are responsible for the highly variable andextremely low bioavailability of the active components.

In one embodiment, the present invention provides a compositioncomprising active ingredients in Red clover, which are optimized toreduce the rate of bone loss and severity of other climactericconditions in postmenopausal women. In one embodiment, the compositioncomprises at least 80% of Biochanin A, and no more than 20% ofgenistein. In another embodiment, the composition comprises at least 80%of Biochanin A, and at least 1% of genistein. In one embodiment, thedose ratio of Biochanin A to genistein ranges from 4:1 to 99:1. In oneembodiment, the dose ratio of Biochanin A to genistein ranges from 2:1to 30:1. In another embodiment, the dose ratio of Biochanin A togenistein ranges from 6:1 to 9:1.

In one embodiment, the composition contains at least 80% of Biochanin A,no more than 15% of genistein, and no more than 6% each of formononetinand daidzein. In one embodiment, the composition contains at least 80%of Biochanin A, at least 1% of genistein, and no more than 5% each offormononetin and daidzein. In one embodiment, the dose ratio ofBiochanin A to formononetin ranges from 5:1 to 100:1. In anotherembodiment, the dose ratio of Biochanin A to formononetin ranges from16:1 to 100:1. In another embodiment, the dose ratio of Biochanin A toformononetin ranges from 20:1 to 30:1. In one embodiment, the dose ratioof Biochanin A to daidzein ranges from 5:1 to 90:1. In anotherembodiment, the dose ratio of Biochanin A to daidzein ranges from 16:1to 90:1. In another embodiment, the dose ratio of Biochanin A todaidzein ranges from 18:1 to 30:1.

In one embodiment, the composition comprises a dosage of totalisoflavones ranging from 0.1 to 10 mg.

In one embodiment, the compositions disclosed herein are obtainedthrough synthetic processes, synthetic sources or natural sources.

In one embodiment, the present invention provides dosage forms of thecompositions that will minimize first-pass metabolism, enhance exposureto active ingredients and minimize inter-individual variability.

In one embodiment, the composition is formulated as parenteral dosageforms (such as intramuscular, subcutaneous and intravenous), buccal,sublingual, or other topical dosage forms which include but are notlimited to transdermal and intra-vaginal.

In one embodiment, the composition is formulated in the form ofgranules, injection, powder, solution, suspension, cream, foam,suppositories or capsules.

In one embodiment, the present invention provides methods of using thecompositions disclosed herein for modulating bone remodeling, comprisingthe step of administering the composition to a subject in need thereof.In one embodiment, the composition is formulated as parenteral dosageforms (such as intramuscular, subcutaneous and intravenous), buccal,sublingual, or other topical dosage forms which include but are notlimited to transdermal and intra-vaginal.

In one embodiment, the present invention provides methods of using thecompositions disclosed herein for modulating hot flashes, comprising thestep of administering the composition to a subject in need thereof. Inone embodiment, the composition is formulated as parenteral dosage forms(such as intramuscular, subcutaneous and intravenous), buccal,sublingual, or other topical dosage forms which include but are notlimited to transdermal and intra-vaginal.

In one embodiment, the present invention provides methods of using thecompositions disclosed herein for modulating vaginal atrophy, comprisingthe step of administering the composition to a subject in need thereof.In one embodiment, the composition is formulated as parenteral dosageforms (such as intramuscular, subcutaneous and intravenous), buccal,sublingual, or other topical dosage forms which include but are notlimited to transdermal and intra-vaginal.

In one embodiment, the present invention provides methods of using thecompositions disclosed herein for modulating one or more postmenopausalor climacteric symptoms, including but not limited to, sleepdisturbances, night sweat, vaginal dryness, diaphoresis, and urinarytract symptoms. The methods comprise the step of administering thecomposition to a subject in need thereof. In one embodiment, thecomposition is formulated as parenteral dosage forms (such asintramuscular, subcutaneous and intravenous), buccal, sublingual, orother topical dosage forms which include but are not limited totransdermal and intra-vaginal.

In one embodiment, the present invention also provides methods of usingthe compositions disclosed herein for treating or preventingosteoporosis, comprising the step of administering the composition to asubject in need thereof. In one embodiment, the composition isformulated as parenteral dosage forms (such as intramuscular,subcutaneous and intravenous), buccal, sublingual, or other topicaldosage forms which include but are not limited to transdermal andintra-vaginal.

In one embodiment, the present invention also provides methods of usingthe compositions disclosed herein for treating or preventing hotflashes, comprising the step of administering the composition to asubject in need thereof. In one embodiment, the composition isformulated as parenteral dosage forms (such as intramuscular,subcutaneous and intravenous), buccal, sublingual, or other topicaldosage forms which include but are not limited to transdermal andintra-vaginal.

In one embodiment, the present invention also provides methods of usingthe compositions disclosed herein for treating or preventing vaginalatrophy, comprising the step of administering the composition to asubject in need thereof. In one embodiment, the composition isformulated as parenteral dosage forms (such as intramuscular,subcutaneous and intravenous), buccal, sublingual, or other topicaldosage forms which include but are not limited to transdermal andintra-vaginal.

In one embodiment, the present invention also provides methods of usingthe compositions disclosed herein for treating or preventing one or morepostmenopausal or climacteric symptoms, including but not limited to,sleep disturbances, vaginal dryness, diaphoresis, night sweat, andurinary tract symptoms. The methods comprise the step of administeringthe composition to a subject in need thereof. In one embodiment, thecomposition is formulated as parenteral dosage forms (such asintramuscular, subcutaneous and intravenous), buccal, sublingual, orother topical dosage forms which include but are not limited totransdermal and intra-vaginal.

In one embodiment, the present invention provides a method of modulatingone or more climacteric symptoms in a subject, the method comprises thestep of administering an effective amount of a composition to thesubject in need thereof, and the composition comprises at least 80% ofBiochanin A, at least 1% of genistein, no more than 5% of formononetinand no more than 5% of daidzein.

In one embodiment of the present method, the composition comprises nomore than 15% of genistein.

In one embodiment of the present method, the ratio of Biochanin A andgenistein ranges from 4:1 to 99:1. In another embodiment, the ratio ofBiochanin A and formononetin ranges from 16:1 to 100:1. In anotherembodiment, the ratio of Biochanin A and daidzein ranges from 16:1 to90:1.

In one embodiment of the present method, the composition comprises adosage of total isoflavones ranging from 0.1 to 10 mg.

In one embodiment of the present method, the composition is administeredvia a route to avoid first-pass gastrointestinal and hepatic effects,and to avoid colonic bacterial metabolism.

In one embodiment of the present method, the composition is formulatedas parenteral, buccal, sublingual, topical, transdermal or intra-vaginaldosage forms. In another embodiment, the composition is formulated as adosage form for intramuscular, subcutaneous or intravenousadministration. In another embodiment, the composition is formulated inthe form of suppository, cream, injection, solution, or suspension.

In one embodiment of the present method, the climacteric symptomscomprise the following:

i) bone remodeling;

ii) bone loss;

iii) osteoporosis;

iv) hot flashes;

v) vaginal atrophy;

vi) vaginal dryness;

vii) diaphoresis;

viii) night sweat;

ix) urinary tract symptoms; and

x) sleep disturbance.

In one embodiment, the present invention provides a composition ofisoflavones comprising at least 80% of Biochanin A, at least 1% ofgenistein, no more than 5% of formononetin and no more than 5% ofdaidzein.

In one embodiment of the present composition, the composition comprisesno more than 15% of genistein.

In one embodiment of the present composition, the ratio of Biochanin Aand genistein ranges from 4:1 to 99:1. In another embodiment, the ratioof Biochanin A and formononetin ranges from 16:1 to 100:1. In anotherembodiment, the ratio of Biochanin A and daidzein ranges from 16:1 to90:1.

In one embodiment of the present composition, the composition comprisesa dosage of total isoflavones ranging from 0.1 to 10 mg.

In one embodiment of the present composition, the composition isformulated as parenteral, buccal, sublingual, topical, transdermal orintra-vaginal dosage forms. In another embodiment, the composition isformulated as a dosage form for intramuscular, subcutaneous orintravenous administration. In another embodiment, the composition isformulated in the form of suppository, cream, injection, solution, orsuspension.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrative,and are not meant to limit the invention as described herein, which isdefined by the claims which follow thereafter.

Throughout this application, various references or publications arecited. Disclosures of these references or publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art to which thisinvention pertains. It is to be noted that the transitional term“comprising”, which is synonymous with “including”, “containing” or“characterized by”, is inclusive or open-ended and does not excludeadditional, un-recited elements or method steps.

Example 1

The objective of this study is to track the events that occur in thelumen of the gastrointestinal tract. The goals are to identify thestability of Red clover components, their physical and enzymaticstability, solubility and absorbability.

Twenty-five red clover extracts containing a diverse composition ofBiochanin A, formononetin, Genistein, Daidzein and their glucosides,along with other minute quantities of coumestrols and lignans have beenprepared either using solvent extraction or a variety of cultivars. Inone embodiment, the aerial portion of red clovers, leaves, stems orleaves and stems, were dried powdered. The plant material was extractedwith 50% ethanol at 50° C. for 1 hour. The resultant sample wascentrifuged and the ethanolic component was removed and dried.

A chromatographic analysis showed that the major ingredients in theseextracts are the glucosides of formononetin and Biochanin A and theirrespective aglycones (FIG. 1). Tiny amounts of genistein, daidzein andtheir glycosides were also found. These data are consistent with what isreported in the literature (Krenn, Unterrieder et al. 2002).

A study of the stability of the key components of a Red clover extractin artificial gastric and intestinal juice showed that the glucosideswere partially (<25%) converted to their respective aglycones.

According to the literature, formononetin and Biochanin A arede-methylated by the intestinal micro flora to produce two activemetabolites daidzein and genistein, respectively (Hur and Rafii 2000).However, the importance of this metabolic pathway at this site isquestioned (Tolleson, Doerge et al. 2002). To understand the relativeimportance of fecal metabolism, the metabolic rate of red cloverphytoestrogens was measured.

Fresh human fecal samples were collected from 4 volunteers. Five gramsof each were pooled together and mixed well with 30 mL BHI culturemedium. The fecal suspension was centrifuged at 200 g for 5 min andsupernatant was decanted and centrifuged at 5,000 g for another 30 min.The resultant precipitate was re-suspended with 10 mL BHI medium toproduce intestinal micro flora solution.

As the biotransformation of drugs by human intestinal bacteria wasdetermined in a 5 mL incubation system containing 250 μL intestinalmicroflora solution, 50 μL stock solution in DMSO in BHI medium. Theincubation system was anaerobically incubated at 37° C. in a GasPak™ EZAnaerobe Pouch System for 0 h, 24 h, 72 h, and 120 h for red cloverisoflavones (the final concentrations for Biochanin A, daidzein, equol,formononetin, and genistein were 100 μM each). Zero-minute incubationsserved as controls. Reactions were stopped by extracting samples with 15ml of ethanol twice. The two ethanol extractions were combined, driedand re-suspended in 80% methanol for HPLC/MS analysis.

Red clover isoflavones are shown to be metabolized extensively by humanintestinal microflora (FIG. 2). When Biochanin A was incubated withintestinal microflora, dihydrobiochanin A, genistein, anddihydrogenistein were formed. Daidzein was metabolized intodihydrodaidzein, and equol. Equol was the most resistant tobio-transformation. At the end of 120 hours, there was still over 60% ofequol left in the incubation media, while there were less than 5% ofBiochanin A, daidzein, and genistein left. The bio-transformed productsof equol were not identified in this study. Formononetin wasbiotransformed into dihydroformononetin, dihydrodaidzein, daidzein, andequol. At the end of 120-hour incubation, there was over 20% offormononetin remained. Genistein was bio-transformed intodihydrogenistein.

This set of studies clearly showed that extensive Phase I metabolismoccurs in the lower part of the intestinal lumen.

Red clover extracts were subjected to permeability measurements usingCaco-2 and MDCK cells. Permeability across these barriers provides anindication of absorbability.

The permeability values of formononetin, Biochanin A, daidzein andgenistein are quite high, suggesting that these components are highlyabsorbable (Table 1). Equol has also been shown to be absorbable.However, the glucosides of the aglycones such as Biochanin A glucosideand ononin have poor permeability suggesting the bioavailability of thesugar conjugates are poorly absorbed. These results are consistent withthat reported in the literature in that when these glycosides areadministered to either animals or humans, no glycosides could bedetected in the blood stream (Setchell, Brown et al. 2002).

TABLE 1 CaCo-2 permeability of isoflavone in a Red clover extractIsoflavones Mean Peff, cm/sec STDEV Biochanin A 1.64E−08 1.11E−09glucoside Biochanin A 1.08E−05 3.83E−07 Daidzein 2.66E−05 1.11E−06Daidzin 5.36E−07 8.30E−08 Formononetin 2.20E−05 6.85E−07 Genistein2.75E−05 1.22E−06 Genistin 3.46E−07 9.20E−08 Ononin 1.22E−07 1.93E−08

The results from the permeability study show that it would be beneficialto convert all the glucosides to their respective aglycones. Twoadvantages of adopting this strategy: a. the variability in the rate andextent of conversion from glucosides to aglycones between subjects willbe removed. A more consistent pattern of aglycone absorption isanticipated. b. dosage calculation for the bioactives will be reduced tothe aglycones only. This simplifies the standardization process.

An optimal extract of Red clover should consist of the aglycones only.An enzymatic or chemical conversion of the glucosides to theirrespective aglycones prior to extraction will be desirable. This can beaccomplished using the literature methods (Tsao, Papadopoulos et al.2006).

Example 2

The objectives of this example are to evaluate gut and liver metabolismof Biochanin A, formononetin, daidzein, genistein and equol. Parametersobtained from these studies are used for estimating the pharmacokineticsof these five components.

Human liver microsomes, intestinal microsomes, and hepatocytes of humanfemale origin were purchased from XenoTech. All chemicals were purchasedfrom Sigma-Aldrich. Isoflavones (biochanin A, daidzein, equol,formononetin, and genistein) were first dissolved in DNSO and then mixedaccording to a randomized table, consisting of 60 samples. The finalDMSO in buffer or media was kept at 0.1%. Protocols supplied by XenoTechInc., the supplier, were used for glucuronidation with microsomalincubation, and hepatocyte incubation. Samples were analyzed usingLC/MS.

FIG. 3 shows that metabolism of the mixtures by human intestinalmicrosomes: Biochanin A (5.41E-4 ml/min/mg protein)>genistein (4.28E-4ml/min/mg protein)>equol (1.07E-4 ml/min/mg protein)>daidzein (6.32E-5ml/min/mg protein)>formononetin (7.31E-5 ml/min/mg protein).

FIG. 4 shows the rate of metabolism of the mixtures by humanhepatocytes. The rates are: equol (1.21E-5 ml/min/millioncells)>biochanin A (8.88E-6 ml/min/million cells)>genistein (5.14E-6ml/min/million cells)>daidzein (4.07E-6 ml/min/millioncells)>formononetin (3.45E-6 mil/min/million cells).

From these studies, it is clearly shown that there are no metabolicinteractions between the five components. In these metabolic studies, noPhase I metabolites were detected suggesting that the formation of PhaseI metabolites, such as daidzein and genistein are formed in theintestinal lumen (Example 1). This piece of information is important inthat the rate of formation of these metabolites is dependent on thesolubility of formononetin and Biochanin A. These results are consistentwith that reported by Howes et al (2002) in that the peak time of thePhase I metabolites is delayed.

Example 3

The objective of this study is to evaluate topical bioavailability ofBiochanin A, genistein, daidzein and formononetin in silico(www.cdc.gov/niosh/topics/skin/finiteskinpermcalc). Values of vaporpressure required for the calculations were obtained from websitewww.thegoodscentscompany.com/episys/ep1504451 for formononetin, websitewww.thegoodscentscompany.com/episys/ep1223481 for biochanin A, websitehuikephytopharm.en.hisupplier.com/product-1881573-Factory-Supply-Natural-Soybean-Extract-Daidzeinfor daidzein and website www.lookchem.com/Genistein/for genistein. Thefollowing are the estimated permeability and bioavailability values ofthese four isoflavones:

TABLE 2 Topical permeability and bioavailability of formononetin,Biochanin A, daidzein and genistein Permeability cm/s Bioavailability %Formononetin −6.42 96.49 Biochanin A −6.34 98.93 Daidzein −6.58 63.75(36.16)* Genistein −6.52 54.02 (45.92)* *Value in bracket is thepercentage absorbed into stratum corneum

In general, the absorption of biochanin A and formononetin ispractically complete. The absorption values of genistein and daidzeinare 64% and 54%, respectively. Provided a proper dosage form isdesigned, the bioavailability of biochanin A and formononetin could becomplete and that of genistein and daidzein are 50% and 60%,respectively.

Example 4

The objectives of this study are to evaluate the effects of individualisoflavones of Red clover and their combinations on osteoblast andosteoclast differentiation.

Materials and Methods

Effects of isoflavones on the differentiation of osteoblast in MC3T3cells followed Ge et al., 2006 and osteoclast differentiation inRaw264.7 cells followed Garcia Palacios et al., 2005. Cell numbers weremeasured with CellTag from Li-Cor Biosciences. Both activities ofalkaline and acid phosphatase were measured with a plate reader at 405nm.

Isoflavones were first dissolved in DMSO and stock solutions wereprepared at 10 mM and the final concentration of total isoflavones intest solution was 10 μM.

Osteoblast and osteoclast differentiations were quantified by measuringactivities of alkaline phosphatase (ALP) and acidic phosphatase (ACP).ALP is highly expressed by the mature osteoblasts and ACP is expressedby osteoclasts. Values of integrated intensity of fluorescence fromCelltag staining serve as a correction factor for the difference in cellnumbers. Therefore, ALP/cell number and ACP/cell number ratios are usedto quantify osteoblast and osteoclast activities.

Results

Osteoblast Differentiation

Confluence MC3T3 cells were treated with 10 μM of isoflavones for 1 weekand then the activity of alkaline phosphatase (ALP) was measured as anindicator of differentiation. Although the difference among isoflavonetreatments was not significant, cells treated with Biochanin Aconsistently showed the highest ALP activity (FIG. 5). To examine ifthere were any synergistic effects, two isoflavones were mixed in a 1:9ratio and tested in the final concentration of 10 μM. Mixtures withhigher ratios (90%) of biochanin A were usually more effective inenhancing the osteoblast differentiation of MC3T3 cells than individualisoflavones alone or their combinations. In the example shown here cellstreated with daidzein:Biochanin A (1:9) and genistein:Biochanin A (1:9)had higher ALP activities than Biochanin A alone (FIG. 6). To examinethe effect of genistein:Biochanin ratio on osteoblast differentiation,genistein was mixed with an increased concentration of Biochanin A (anincrement of 10%). With the increased concentration of Biochanin A, thedifferentiation enhancing ability of the mixture increased and thendropped off when the mixture only contained Biochanin A (FIG. 7).

Osteoclast Differentiation

Raw246.7 cells were treated with MCSF and RANKL to stimulate thedifferentiation of osteoclasts. Isoflavones were added at the finalconcentration of 10 μM to examine their ability to inhibitdifferentiation. Cells treated with Biochanin A and mixtures ofBiochanin A and genistein, daidzein or formononetin at a 9:1 ratioshowed the highest inhibition in osteoclast differentiation (FIG. 8). Itis also noted that pure genistein and daidzein have the opposite effectsin stimulating osteoclast differentiation as compared to the inhibitingeffects observed in pure Biochanin A.

CONCLUSIONS

Contrary to its low estrogenicity (Beck, Unterrieder et al. 2003),Biochanin A is found to be most effective in enhancing thedifferentiation of osteoblast and to inhibit the differentiation ofosteoclasts. Mixtures of Red clover aglycones containing highproportions of Biochanin A show synergistic effects. In one embodiment,the preferred ratios of the components at the site of action are 80 to90% of Biochanin A, up to 15% genistein, and no more than 10% each ofdaidzein and formononetin.

The 9:1 concentration ratio of Biochanin A to genistein, daidzein orformononetin described herein is believed to provide desired“estrogen-like” activity that could modulate or prevent climacteric orpostmenopausal symptoms. The beneficial effects of this 9:1 ratio arethus not limited to the effects on bone cells or bone-relatedconditions, but also on symptoms or conditions directly or indirectlyrelated to estrogens.

Example 5

The protocol used by (Moon, Sagawa et al. 2006) was used for measuringplasma protein binding of the absorbable aglycones. Parameters have beenused for PBPK simulation. Conjugates of Biochanin A, formononetin,Genistein, Daidzein and Equol are predominant components in plasma. Therespective aglycones constitute less than 5% of the total concentration.Plasma protein binding of biochanin A, formononetin, genistein and equolare over 97% and daidzein was approximately 80%.

Example 6

The objectives of this study are: 1. To evaluate solubility interactionsamong the four phytoestrogens, which are native to Red clover, namely,Biochanin A, formononetin, daidzein and genistein; 2. To evaluatepotential differences in physicochemical properties of extractscontaining the same amounts of phytoestrogens.

Materials and Methods

Interactions Among Isoflavones

Isoflavones: daidzein, genistein, formononetin and Biochanin A wereobtained from Indofine. Simulated intestinal fluid buffer powdermimicking a fasted state (FaSSIF) was obtained from Biorelevant SIFmedia, Biorelevant.com, Switzerland.

To prepare accurate concentrations of isoflavones in microtubes, stocksolutions of individual isoflavones were prepared at 1 mg/mL inmethanol. The amount of isoflavone designated to be held constant wasprovided at a concentration that well exceeded (about 20×) thesaturation concentration for that isoflavone. The appropriate amount ofstock was transferred to each microtube and the material was dried downin a vacuum centrifuge.

In the case of Biochanin A competition with other flavones (daidzein,genistein and formononetin), the amount of Biochanin A was held constantat 200 μg in 1 ml buffer (saturation concentration for Biochanin A inFaSSIF is about 8 μg/mL). The competing isoflavone was prepared at 0, 1,5, 10, 50, 100, 200 μg/mL (FIGS. 11 and 12) and, in the case offormononetin, 400 μg/mL (FIG. 9).

In the case of formononetin competition with Biochanin A, the amount offormononetin was held constant at 50 μg in 1 ml buffer (saturationconcentration for formononetin in FaSSIF is about 2 μg/mL). Thecompeting Biochanin A was prepared at 0, 1, 5, 10, 50, 100 and 200 μg/mL(FIG. 10).

Each tube was then reconstituted with 1 mL of FaSSIF buffer, sonicatedand allowed to equilibrate with occasional agitation for 24 hours at 37°C. This produced a solution that contained saturated isoflavoneconcentrations mimicking mammalian intestinal conditions.

At the end of 24 hours each tube containing isoflavones and FaSSIFbuffer was briefly centrifuged at 5000 rpm in a microcentrifuge held at37° C. (2 minutes). A portion of the supernatant (400 μL) was thenimmediately placed in a centrifugal filter unit (UltraFree-MC-GV 0.22μM) and the sample filtered by centrifugation (8000 rpm, 5 minutes, 37°C.). Upon filtration 200 μL filtrate was immediately placed in amicrotube and 200 μL methanol added to ensure that the isoflavonesremained in solution. The sample was mixed and 200 μL of the mixture wastransferred to injection vials provided with 200 μL polypropyleneinjection inserts.

The samples were analyzed by HPLC with diode array detection at 260 nmusing 20 μL injections.

Results

When Biochanin A was placed in media representing fasted digestive juiceat a concentration of 200 μg/mL at 37° C. (an amount about 25 times thesoluble saturation value) the amount in solution was determined to beabout 7.1 μg/mL (FIG. 9). As formononetin is introduced, Biochanin Asaturation concentration dropped in a dose dependent manner and wasreduced to about 4.5 μg/mL in the presence of 400 μg/mL formononetin(FIG. 9).

When the experiment was done holding formononetin at 50 μg/mL (about 25times formononetin's saturation solubility in fasted media), increasingconcentrations of Biochanin A did not affect the saturationconcentration of Formononetin (FIG. 10). It is concluded that thepresence of Biochanin A does not influence the saturation concentrationof the much less soluble formononetin.

In experiments where Biochanin A solubility was investigated in thepresence of varying concentrations of genistein, a different set ofresults was obtained. When Biochanin A is placed in media representingfasted digestive juice at a concentration of 200 μg/mL at 37° C. theamount in solution was determined to be about 10.8 μg/mL (FIG. 11). Asgenistein was introduced, Biochanin A saturation concentration increasedin a dose dependent manner and reached about 24.2 μg/mL in the presenceof 200 μg/mL genistein (FIG. 11). It is concluded that the solubility ofBiochanin A is enhanced by the presence of genistein in fasted digestivemedium.

In experiments where Biochanin A saturation solubility was investigatedin the presence of varying concentrations of daidzein, a set of resultsdifferent from both the formononetin and genistein experiments wereobtained. When Biochanin A was placed in media representing fasteddigestive juice at a concentration of 200 μg/mL at 37° C. the amount insolution was determined to be about 10.0 μg/mL (FIG. 12). As daidzeinwas introduced, the Biochanin A saturation concentration remainsunaffected and was about 8.7 μg/mL in the presence of 200 μg/mL daidzein(FIG. 12). It is concluded that the solubility of biochanin A isunaffected by the presence of daidzein in fasted digestive media.

This set of studies clearly showed that interactions among isoflavonesare not predictable. The solubility of Biochanin A in simulatedintestine juice is reduced by formononetin, enhanced by genistein, andnot affected by daidzein.

Similar results are obtained when simulated intestinal juice mimickingthe fed state was used (data not shown).

Solubility of Isoflavones in Red Clover Extracts

Two Red clover extracts containing 40% total isoflavones were examined:Shaanix Tianzun BN 078201205123 and Acetar TYR081023.

One gram extract was placed in a disposable 12 mL glass screw top testtube. 10 mL of 100% methanol was added and the tube was capped. The tubewas mixed and placed in an ultrasonic water bath for 5 minutes. The tubewas then shaken every 15 minutes for 1 hour. Mixing was done at roomtemperature. At the end of the 1-hour incubation the tube wascentrifuged (Eppendorf 5804 R, 1500 rpm, 10 minutes) and the supernatantwas collected and set aside. Another 10 mL of methanol was introduced tothe tube on top of the sediment and the material was sonicated, mixedand incubated as described above. This process was repeated such that 12washes from the material were collected. The precipitate from the finalwash was re-suspended in methanol.

A 100 μL aliquot collected from each 10 mL wash was diluted 1:10 with80% methanol, centrifuged and the supernatant analyzed by HPLC withdiode array detection at 260 nm using 5 or 20 μL injections. 20 μLinjections were used for later washes (wash 6-12) in which daidzein,genistein and Biochanin A concentrations were much lowered.

The remaining wash supernatants as well as the re-suspended finalresidue were individually dried down in pre-weighed microtubes toprovide an estimate of solid weight recovered in each wash.

An estimate of isoflavone concentrations in the original extracts wasmade by dissolving the extracts at 1 mg/mL in 80% methanol with warmingand sonication. An aliquot was diluted 1:10 with 80% methanol andcentrifuged. A 5 μL injection was analyzed by HPLC.

The two 40% isoflavone products extract (Acetar and Shaanxi) are foundto be different from each other. There was a difference in appearancebetween the two 40% extracts as one has a dark green-gray color (Acetar)and the other one is off white (Shaanxi) after extraction with methanol.

The Acetar extract did not release isoflavones as rapidly when comparedto that of Shaanix (Comparing FIGS. 13 and 14). Biochanin A was stillbeing extracted after 12 sequential extracts. It appears that somethingin the Acetar extract is binding the isoflavones and only slowlyreleasing them into the methanol (FIG. 14).

The results of this study clearly showed that isoflavones from differentRed clover extracts produced using different procedures could havevastly different solubility. Since absorption of isoflavones is highlydependent on their solubility, isoflavones prepared from sources ormaterials with identical labels may have different bioavailability. Thismay in part explain the inconsistent clinical results reported in theliterature (Booth, Piersen et al. 2006).

The hypothesis that solubility may be an issue of phytoestrogenabsorption was tested by examining the dissolution profile of acommercially available Red clover product, PROMENSIL® (30 tablets in abox, Lot # [B] 48449, Exp. March 2011).

FIG. 15 shows that the dissolution of phytoestrogens in the product isnot complete, lending evidence to support the idea that aninappropriately formulated product will perform erratically because ofabsorption issues. It should also be pointed out that the phytoestrogensin PROMENSIL® consist of both aglycones and their glucosides.Compounding the bioavailability issue, both of these species are notcompletely dissolved under the experimental condition studied.

Example 7

One of the objectives of this example is to employ the proprietarypharmacodynamic/pharmacokinetic (PBPK) model to simulate thepharmacokinetic behavior of the active phytoestrogens in Red clover.Another objective of this example is to design effective formula anddosage forms of phytoestrogen products based on the simulation resultsand in vitro studies of the present invention.

Simulation Using Proprietary PBPK Model

Results from Examples 1, 2 and 5 are used as inputs into the proprietaryPBPK model to simulate plasma concentration profiles of the fourphytoestrogens: Biochanin A, formononetin, daidzein and genistein andtheir Phase II metabolites.

Using the parameters generated in Examples 1, 2 and 5, the proprietaryPBPK model was adapted to describe the pharmacokinetics of Red cloverisoflavones. The proprietary PBPK model was first validated using theclinical data of Howes et al. (2002). The model was considered validatedwhen the simulated results of Area Under the Curve (AUC) values of thePhase II metabolites are agreeable (within 2-fold, Table 3, second andthird column) with that published by Howes et al. (2002).

After the validation of the proprietary PBPK model, the resultant PKdata were used to estimate other PK data, dose and routes ofadministration. In one embodiment, the PBPK model was used to simulatethe AUC values of isoflavones after oral and intravenous administration(Table 3, fourth and fifth column).

TABLE 3 Comparison of plasma levels of isoflavones obtained fromclinical samples and simulation results using the PBPK model AUC₀₋₂₄,ng*h/mL Estimated Parameters Phase II metabolites Aglycones Howes'Aglycones (simulation) Bioavailability Clearance data^(#) SimulationOral IV % ml/min Formononetin 112 ± 35 123 9.47 1989 0.48 268 BiochaninA  518 ± 518 519 4.99 2422 0.21 337 Daidzein  891 ± 135 693 7.49 2612.87 192 Genistein 1463 ± 115 1231 3.85 229 1.68 218 ^(#)Data reportedby Howes et al. (2002). Two Promensil tablets contain 32 mg ofFormononetin, 49 mg of Biochanin A, 3 mg of daidzein and 3 mg ofgenistein. Based on the maximum plasma concentration (Cmax) in ng/mLreported by Howes et al. (2002), the maximum plasma concentration in μMwas calculated: 0.042 μM for Formononetin, 0.168 μM for Biochanin A,0.423 μM for daidzein and 0.247 μM for daidzein.

Clinical data of Howes et al. (2002) indicated that the plasmaisoflavone levels in humans are highly varied (See C. of Table 1 ofHowes et al.). The huge variation of plasma isoflavone levels observedcould be due to high first-pass metabolism, lack of solubility andsolubility interactions among isoflavones. The solubility issues wouldpose an upper limit of isoflavone absorption, preventing the achievementof a higher plasma concentration.

Howes et al's (2002) data also showed that AUC values among subjects arehighly variable (>10 fold). This variation can be explained by theinstability of glucosides, low solubility of the aglycones, interactionamong aglycones at the solubility level, aglycone metabolism of theaglycones by colonic bacteria and high first-pass gut and livermetabolism.

Limited solubility of phytoestrogens in the small intestine may beresponsible for the lack of a dose-dependent increase in clinicalresponse to Red clover isoflavones (Booth, Piersen et al. 2006).

Comparing the AUC values of phytoestrogens obtained after intravenousadministration and oral administration, it is observed that theestimated AUC values of aglycone obtained after intravenousadministration is approximately 35 to 500 times higher than those afteroral administration (Table 3, fourth and fifth column).

This simulation suggests that bioavailability of phytoestrogens could beenhanced by administering the compounds via non-oral route. For example,after intravenous administration, AUC of phytoestrogens could be 35 to500-fold higher than that of a comparative oral dose (Table 3). Thissuggests that non-oral dosage could be as low as 0.2 to 3% of that oforal doses.

High variability, low bioavailability and solubility limited absorptionare the major causes for therapeutic failure. In a recent review (Lagariand Levis 2014), it was reported that there was a much higher proportionof clinical trials showing Red clover phytoestrogens were ineffective intreating postmenopausal bone loss and climacteric symptoms than theeffective ones. Dosages used for clinical trials went as high as 80 mgtotal phytoestrogens. Based on the literature reports and observationsmade in this invention, plasma concentrations of biochanin A andgenistein measured by Howes et al. (2002) are considered to bemarginally effective since the total phytoestrogen dose used by Howe etal was 87 mg.

Based on the results described in this invention, failure of Red clovertherapy is not surprising because low solubility, high first-passeffects and variable colonic metabolism are key factors which lead tolow bioavailability and high inter-individual variability of thephytoestrogens.

The dosages of total phytoestrogens used in clinical trials aretypically as high as 80 mg but could only achieve low AUC values of theisoflavones, hence limiting the therapeutic efficacy of the products.The present simulation data (not shown) indicates that AUC of individualisoflavone would not increase with the increasing oral dose because ofthe limited solubility and first-pass metabolism. That is, dosageshigher than 80 mg are not likely to further enhance the therapeuticefficacy. These results are consistent with that reported by Booth et.al. (2006), who showed that the efficacy of phytoestrogens was notdose-dependent.

Hence, to deal with the limited efficacy of phytoestrogen products, oneof the objectives of the present invention is to account for thepharmacokinetic properties of the isoflavones and to design effectiveisoflavone products with optimal dosage forms and dose ratios of theisoflavones.

Table 4 shows one example of how the present invention designs a Redclover composition for topical administration based on the simulationresults, that is, in view of the pharmacokinetic and the pharmacodynamicproperties of the components. In this example, the target AUC to beachieved for biochanin A by the topically administered Red clovercomposition is defined at 10 times of the AUC value obtained after theoral administration of the composition (AUC_(Oral)). The target AUCratios for biochanin A: genistein is 9:1, biochanin A: formononetin is20:1 and biochanin A: daidzein is 20:1. For each isoflavone, the topicaldose is calculated based on the target AUC value as calculated and theAUC value obtained after intravenous administration as simulated(AUC_(IV)). Since AUC_(IV) takes into account of 100% absorption of thecomponent and clearance from the body, a calculation of topical dosebased on AUC_(IV) would provide a good estimation if the component is100% absorbed after administration, otherwise normalization is requiredto account for the lesser extent of absorption. The present simulationresults showed that Biochanin A and formononetin are 100% absorbed whilegenistein and daidzein are 54.02 and 63.75%, respectively (Table 2).Hence, the estimated topical dose of formononetin, daidzein andgenistein were calculated based on specified ratios as inferred from thesimulation data (Table 4).

TABLE 4 Extrapolated topical doses of four isoflavones and their AUCAUC₀₋₂₄, ng*h/mL Aglycones % of total Dose ratio of (simulation)Molecular Target AUC Topical** topical Biochanin A: Oral IV weight(ng*h/mL)* dose (mg) dose isoflavone Biochanin A 4.99 2422 284.26 49.91.01 82.13 1 Formononetin 9.47 1989 268.26 2.50 0.04 3.27 25.15 Daidzein7.49 261 254.33 2.50 0.045 3.66 22.44 Genistein 3.85 229 270.241 5.540.134 10.94 7.51 Total dose 1.23 100 / *For Biochanin A, the target AUCis 10 times the AUC value of orally taken Biochanin A. Target AUC offormononetin, daidzein and genistein were calculated based on thefollowing ratios - Biochanin A:formononetin (20:1); BiochaninA:genistein(9:1); and Biochanin A:daidzein (20:1). **Dosages could be aslow as estimated assuming optimal absorption.

The data in Table 4 shows that when the composition is formulated fortopical administration, the total dosage of phytoestrogens could be aslow as 1.23 mg, which is extremely low compared to the common dosage 80mg for oral administration. The topical dosage form minimizes theproblems of limited solubility and first-pass metabolism, and hencesignificantly enhances the bioavailability and therapeutic efficacy ofthe products.

In the example provided by Table 4, the dose ratio of Biochanin A toformononetin, daidzein and genistein are about 25:1, 22:1 and 8:1respectively. Hence, in one embodiment, the present composition isformulated to include the four isoflavones in the following ranges ofratios: Biochanin A to formononetin of about 20:1 to 30:1, Biochanin Ato daidzein of about 18:1 to 30:1, and Biochanin A to genistein of about6:1 to 9:1.

In one embodiment, doses of components to be administered to a subjectare adjusted not only in view of the pharmacokinetic and pharmacodynamicproperties of the components but also in a way that accounts for anyvariability in the simulation data in order to achieve a desired ratioof components at the site of action. In the present study, a two-foldvariability in the results of simulation is expected and taken intoaccount for designing the formulation of the present compositions.Therefore, based on the formulation in Table 4, the dose of the fourisoflavones and their dose ratios can be translated to other doses andratios accounting for the two-fold variation in the clinical data and inthe simulation (See Table 5). In one embodiment, the dose ratio ofBiochanin A to genistein is extrapolated to about 2:1 to 30:1. Inanother embodiment, the dose ratio of Biochanin A to formononetin isextrapolated to about 5:1 to 100:1. In another embodiment, the doseratio of Biochanin A to daidzein is extrapolated to about 5:1 to 90:1.

In one embodiment of the present composition which comprises at least80% of Biochanin A, at least 1% of genistein, no more than 5% offormononetin and no more than 5% of daidzein, the ratios of isoflavonesare: about 4:1 to 99:1 of Biochanin A to genistein, about 16:1 to 100:1of Biochanin A to formononetin, and about 16:1 to 90:1 of Biochanin A todaidzein. Dose ratios for other doses of isoflavones can be deducedsimilarly.

TABLE 5 Extrapolation of topical doses and dose ratios of fourisoflavones Range Range of of target dose of Dose ratio of Target AUCAUC* isoflavones Biochanin A: (ng * h/mL) (ng * h/mL) (mg) isoflavone⁺Biochanin A 49.9 24.95-99.80 0.50-2.02 1 Formononetin 2.50 1.25-4.990.02-0.08  6.29-100.60 Daidzein 2.50 1.25-4.99 0.022-0.090 5.61-90.00Genistein 5.54  2.77-11.09 0.07-0.27 1.88-30.03 *Range of target AUC istaken as 50-200% of the target AUC value. ⁺Lower limit of dose ratio =lowest dose of Biochanin A/highest dose of isoflavone; Upper limit ofdose ratio = highest dose of Biochanin A/lowest dose of isoflavone.Consideration of In Vitro Efficacy and Interactions Among Isoflavones

As discussed in Example 4, the present invention discovered that theconcentration ratio 9:1 of isoflavones is effective in modulatingestrogen-related activity. The concentration of isoflavones used in thepresent in vitro studies was 10 μM. This concentration rangesapproximately 20 times for genistein (10 μM/0.423 μM) to 240 times forformononetin (10 μM/0.042 μM) higher than that observed in humans (Howeset al., 2002). This difference is to be expected as the duration ofstudy in vitro (one week) was a lot shorter than that in the clinicaltrials (>6 months) (Booth, 2006).

Results from the present in vitro studies (Example 4) suggest that invivo plasma concentration ratio of biochanin A to genistein of 9:1provides the highest efficacy. It should be noted that the ratio of thecomponents, rather than the actual concentrations used in the in vitrostudies, is more clinically relevant. In one embodiment, the 9:1 ratioof biochanin A to genistein used in Example 4 is translated to a doseratio accounting for the higher clearance rate of biochanin A thangenistein, and in view of the simulation data obtained from theproprietary PBPK model.

Commercially, it is common to find Red clover extracts containing higherproportions of formononetin. As demonstrated in the present invention,Biochanin A is found to be the most effective in modulating thedifferentiation of bone cells (Example 4) and formononetin is shown tolower Biochanin A's solubility in simulated intestinal fluid (Example6). Thus it is not surprising to find that Red clover extractscontaining high formononetin are ineffective.

In the realm of solubility limitations, an effective combination of Redclover phytoestrogens should contain very low levels of Formononetin (<2to 10%).

Genistein has been found to have dual functions. It enhances thesolubility of Biochanin A and acts synergistically with Biochanin A inenhancing bone remodeling.

A low percentage of daidzein and formononetin, approximately 10% of thatof biochanin A (FIG. 8) has also been found to have synergisticantiresorptive effects.

Taking together, an ideal combination of Red clover phytoestrogensshould contain a high content of Biochanin A (>80%) and smaller contentsof genistein, daidzein and formononetin. In one embodiment, the doseratios of these components can be adjusted in view of the abovesimulation data and/or the in vitro studies in Example 4.

To avoid extensive first-pass effects, solubility issues and variablecolonic metabolism, an alternative route of administration other thanoral should be employed. The present composition is formulated foradministration via different routes in accordance with the need. In oneembodiment, the present composition is formulated for administration viathe following routes: parenteral such as intravenous, subcutaneous andintramuscular. In another embodiment, the present composition isformulated for administration via topical, transdermal, vaginal, buccalor sublingual administration.

In one embodiment, the dosage of phytoestrogens can be less than 0.2-10%of the normal clinical dose, which is about 80 mg. That is, the dosageof phytoestrogens used herein can be less than 0.16 to 8 mg. These lowdosages should provide significantly higher plasma levels of BiochaninA, genistein, daidzein and formononetin when compared to a regular 80 mgdose of Red clover isoflavones.

Assuming an 80 mg dose is marginally active (Lagari and Levis 2014), thecombination of phytoestrogens as disclosed herein would greatly enhanceclinical effectiveness of Red clover phytoestrogens in treating boneloss and other climacteric symptoms.

Example 8

This example records the experience of two postmenopausal women aftertaking a vaginal suppository designed based on the present invention fora period 7 to 14 days. The first woman was a 55-year-old postmenopausalfemale who suffered from hot flashes and vaginal atrophy, but otherwisehealthy. The second woman is a 64-year-old postmenopausal woman whosuffered from vaginal dryness and osteoarthritis.

Vaginal suppositories containing 5 mg of biochanin A, 0.104 mg ofgenistein, 0.087 mg of formononetin and 0.071 mg of daidzein wereprepared using a blend of polyethylene glycol (PEG) base, containing 40%PEG 300 and 60% PEG 3350 and silica gel micro. In this particularformulation, the dose ratios of isoflavones are: about 48:1 of BiochaninA to genistein, about 58:1 of Biochanin A to formononetin, and about70:1 of Biochanin A to daidzein.

The first subject applied a suppository once a day before bedtime forsix consecutive days. After the first dose, the preparation has beenable to arrest the onset of hot flashes and night sweat (feeling ofhotness and heavy sweating) within two hours and the symptoms did notrecur during this six-day period. Vaginal dryness is reported todisappear after the third application.

Using the same dosing regimen, the symptom of vaginal dryness for thesecond woman disappeared on the third day. During the treatment period,vaginal dryness did not reappear.

The two studies empirically proved that the present compositions, whenformulated for vaginal administration and formulated with optimal ratiosof the isoflavones, generated immediate therapeutic effects in a fewdays. Therefore, the present invention represents a big improvement overconventional treatments which usually take several weeks to take effectsand at best generate marginal therapeutic effects.

In summary, the present invention investigated the effects of differentcombination of isoflavones of Red clover on the differentiation ofosteoblasts and osteoclasts (Example 4), studied the solubility andinteractions among these isoflavones (Example 6), and analyzed thepharmacokinetic properties of these isoflavones using a proprietaryphysiologically based pharmacokinetic and pharmacodynamic model (PBPKPD)(Example 7). The present findings provide a basis to design compositionsthat are more effective in modulating climacteric symptoms whilerequiring a much lower dosage of Red clover phytoestrogens thanconventional products available at the time of the invention. Theclinical data in Example 8 proved that the present compositions areeffective in treating climacteric symptoms including hot flashes, nightsweat and vaginal dryness within a few days, and the daily dosage ofphytoestrogens required is only about 6.6% of the conventional dosage 80mg. In support of the data described in this invention, the presentcompositions, when formulated properly, could be used to modulate,prevent or treat climacteric symptoms within a short period of time.

-   Beck, V., U. Rohr and A. Jungbauer (2005). “Phytoestrogens derived    from red clover: an alternative to estrogen replacement therapy?” J    Steroid Biochem Mol Biol 94(5): 499-518.-   Beck, V., E. Unterrieder, L. Krenn, W. Rubelka and A. Jungbauer    (2003). “Comparison of hormonal activity (estrogen, androgen and    progestin) of standardized plant extracts for large scale use in    hormone replacement therapy.” J Steroid Biochem Mol Biol 84(2-3):    259-268.-   Booth, N. L., C. R. Overk, P. Yao, J. E. Burdette, D. Nikolic, S. N.    Chen, J. L. Bolton, R. B. van Breemen, G. F. Pauli and N. R.    Farnsworth (2006). “The chemical and biologic profile of a red    clover (Trifolium pratense L.) phase II clinical extract.” J Altern    Complement Med 12(2): 133-139.-   Booth, N. L., C. E. Piersen, S. Banuvar, S. E. Geller, L. P. Shulman    and N. R. Farnsworth (2006). “Clinical studies of red clover    (Trifolium pratense) dietary supplements in menopause: a literature    review.” Menopause 13(2): 251-264.-   Chen, J., H. Lin and M. Hu (2005). “Absorption and metabolism of    genistein and its five isoflavone analogs in the human intestinal    Caco-2 model.” Cancer Chemother Pharmacol 55(2): 159-169.-   Chen, J., S. Wang, X. Jia, S. Bajimaya, H. Lin, V. H. Tam and M. Hu    (2005). “Disposition of flavonoids via recycling: comparison of    intestinal versus hepatic disposition.” Drug Metab Dispos 33(12):    1777-1784.-   Fernandez, E., S. Gallus, C. Bosetti, S. Franceschi, E. Negri and C.    La Vecchia (2003). “Hormone replacement therapy and cancer risk: a    systematic analysis from a network of case-control studies.” Int J    Cancer 105(3): 408-412.-   Gambacciani, M., M. Ciaponi and A. R. Genazzani (2007). “The HRT    misuse and osteoporosis epidemic: a possible future scenario.”    Climacteric 10(4): 273-275.-   Gambacciani, M., P. Monteleone, A. Sacco and A. R. Genazzani (2003).    “Hormone replacement therapy and endometrial, ovarian and colorectal    cancer.” Best Pract Res Clin Endocrinol Metab 17(1): 139-147.-   Ghazanfarpour, M., R. Latifnejad Roudsari, G. Treglia and R. Sadeghi    (2015). “Topical administration of isoflavones for treatment of    vaginal symptoms in postmenopausal women: A systematic review of    randomised controlled trials.” J Obstet Gynaecol 35(8): 783-787.-   Ghazanfarpour, M., R. Sadeghi, R. Latifnejad Roudsari, K. Mirzaii    Najmabadi, M. Mousavi Bazaz, S. Abdolahian and T. Khadivzadeh    (2015). “Effects of red clover on hot flash and circulating hormone    concentrations in menopausal women: a systematic review and    meta-analysis.” Avicenna J Phytomed 5(6): 498-511.-   Ghazanfarpour, M., R. Sadeghi and R. L. Roudsari (2016). “The    application of soy isoflavones for subjective symptoms and objective    signs of vaginal atrophy in menopause: A systematic review of    randomised controlled trials.” J Obstet Gynaecol 36(2): 160-171.-   Ghazanfarpour, M., R. Sadeghi, R. L. Roudsari, I. Khorsand, T.    Khadivzadeh and B. Muoio (2016). “Red clover for treatment of hot    flashes and menopausal symptoms: A systematic review and    meta-analysis.” J Obstet Gynaecol 36(3): 301-311.-   Howes, J., M. Waring, L. Huang and L. G. Howes (2002). “Long-term    pharmacokinetics of an extract of isoflavones from red clover    (Trifolium pratense).” J Altern Complement Med 8(2): 135-142.-   Hur, H. and F. Rafii (2000). “Biotransformation of the isoflavonoids    biochanin A, formononetin, and glycitein by Eubacterium limosum.”    FEMS Microbiol Lett 192(1): 21-25.-   Jia, X., J. Chen, H. Lin and M. Hu (2004). “Disposition of    flavonoids via enteric recycling: enzyme-transporter coupling    affects metabolism of biochanin A and formononetin and excretion of    their phase II conjugates.” J Pharmacol Exp Ther 310(3): 1103-1113.-   Krenn, L., I. Unterrieder and R. Ruprechter (2002). “Quantification    of isoflavones in red clover by high-performance liquid    chromatography.” J Chromatoqr B Analyt Technol Biomed Life Sci    777(1-2): 123-128.-   Lagari, V. S. and S. Levis (2014). “Phytoestrogens for menopausal    bone loss and climacteric symptoms.” J Steroid Biochem Mol Biol 139:    294-301.-   Liu, J., J. E. Burdette, H. Xu, C. Gu, R. B. van Breemen, K. P.    Bhat, N. Booth, A. I. Constantinou, J. M. Pezzuto, H. H. Fong, N. R.    Farnsworth and J. L. Bolton (2001). “Evaluation of estrogenic    activity of plant extracts for the potential treatment of menopausal    symptoms.” J Agric Food Chem 49(5): 2472-2479.-   Ma, D. F., L. Q. Qin, P. Y. Wang and R. Katoh (2008). “Soy    isoflavone intake inhibits bone resorption and stimulates bone    formation in menopausal women: meta-analysis of randomized    controlled trials.” Eur J Clin Nutr 62(2): 155-161.-   Magee, P. J. (2011). “Is equol production beneficial to health?”    Proc Nutr Soc 70(1): 10-18.-   Moon, Y. J., K. Sagawa, K. Frederick, S. Zhang and M. E. Morris    (2006). “Pharmacokinetics and bioavailability of the isoflavone    biochanin A in rats.” Aaps J 8(3): E433-442.-   Overk, C. R., P. Yao, L. R. Chadwick, D. Nikolic, Y. Sun, M. A.    Cuendet, Y. Deng, A. S. Hedayat, G. F. Pauli, N. R.    Farnsworth, R. B. van Breemen and J. L. Bolton (2005). “Comparison    of the in vitro estrogenic activities of compounds from hops    (Humulus lupulus) and red clover (Trifolium pratense).” J Agric Food    Chem 53(16): 6246-6253.-   Seelig, M. S., B. M. Altura and B. T. Altura (2004). “Benefits and    risks of sex hormone replacement in postmenopausal women.” J Am Coll    Nutr 23(5): 482S-496S.-   Setchell, K. D., N. M. Brown, L. Zimmer-Nechemias, W. T.    Brashear, B. E. Wolfe, A. S. Kirschner and J. E. Heubi (2002).    “Evidence for lack of absorption of soy isoflavone glycosides in    humans, supporting the crucial role of intestinal metabolism for    bioavailability.” Am J Clin Nutr 76(2): 447-453.-   Setchell, K. D. and A. Cassidy (1999). “Dietary isoflavones:    biological effects and relevance to human health.” J Nutr 129(3):    758S-767S.-   Tolleson, N. H., D. R. Doerge, M. I. Churchwell, M. M. Marques    and D. W. Roberts (2002). “Metabolism of biochanin A and    formononetin by human liver microsomes in vitro.” J Agric Food Chem    50(17): 4783-4790.-   Tsao, R., Y. Papadopoulos, R. Yang, J. C. Young and K. McRae (2006).    “Isoflavone profiles of red clovers and their distribution in    different parts harvested at different growing stages.” J. Agric.    Food Chem. 54: 5797-5805.-   Wuttke, W., H. Jarry and D. Seidlova-Wuttke (2007).    “Isoflavones—safe food additives or dangerous drugs?” Ageing Res Rev    6(2): 150-188.

What is claimed is:
 1. A method of modulating one or more climactericsymptoms in a subject, comprising the step of administering to thesubject in need thereof an effective amount of a composition comprisingBiochanin A, genistein, formononetin and daidzein, wherein thecomposition comprises 80%-96% Biochanin A, 1%-12% genistein, 1%-5%formononetin and 1%-5% of daidzein, wherein the effective amountincludes 1.0-6.0 mg of total isoflavones, and the composition isadministered via a route to avoid first-pass gastrointestinal andhepatic effects, and to avoid colonic bacterial metabolism, wherein theclimacteric symptoms are selected from the group consisting of: a. hotflashes; b. vaginal atrophy; c. vaginal dryness; d. diaphoresis; e.night sweat; f. urinary tract symptoms; and g. sleep disturbance.
 2. Themethod of claim 1, wherein the composition includes 5 mg of Biochanin A,0.104 mg of genistein, 0.087 mg of formononetin, and 0.071 mg ofdaidzein.
 3. The method of claim 1, wherein the composition includes aratio of Biochanin A to genistein is about 48:1.
 4. The method of claim1, wherein the composition includes a ratio of Biochanin A toformononetin is about 58:1.
 5. The method of claim 1, wherein thecomposition includes a ratio of Biochanin A to daidzein is about 70:1.6. The method of claim 1, wherein the composition is formulated asparenteral, buccal, sublingual, topical, transdermal or intra-vaginaldosage forms.
 7. The method of claim 1, wherein the composition isformulated as a dosage form for intramuscular, subcutaneous orintravenous administration.
 8. The method of claim 1, wherein thecomposition is formulated in the form of suppository, cream, injection,solution, or suspension.